JP2749615B2 - Novel indicator compounds, their preparation and use of the compounds in iron analysis systems - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention provides a compound represented by the general formula (I) Wherein R ₁ represents hydrogen, halogen or C ₁ -C ₄ alkyl, and R ₂ represents C ₁ -C ₄ alkyl, aryl or heteroaryl, or substituted C ₁ -C ₄ alkyl, aryl or heteroaryl. And a method for producing the compound. These compounds can be used in an iron analysis system.

Various changes in iron metabolism manifest as both deficiency and hyperplasia of this element. Iron deficiency is widespread and obviously affects people in highly developed countries as well as those with malnutrition. Iron deficiency affects women primarily due to the large loss of iron associated with menstruation, jeopardizes pregnant women due to increased iron demand, and also affects patients with ulcers or other acute or chronic bleeding. I'm dying. Newborns are also particularly susceptible to the disease.

Accurate and reproducible laboratory testing methods are needed to diagnose iron deficiency, distinguish between low blood iron and iron deficiency in inflammatory diseases, and monitor treatment.

Laboratory diagnostics in this area essentially utilize measurements of: Blood iron blood transferrin blood ferritin blood cell volume and hemoglobin free erythrocyte protoporphyrin.

The free erythrocyte protoporphyrin test is currently being developed from the experimental stage for routine testing in the clinic, although all other test methods are almost established in the clinic.

As used herein, the term iron in blood refers to the concentration of iron actually carried into the plasma, and thus the concentration of iron bound to transferrin. Although it is not a reliable indicator of the body's iron content, iron ion analysis is valuable for estimating the state of stored iron. The main causes that can cause iron deficiency or hyperplasia are shown in Table 1 below.

Table 1 Causes of reduced iron changes in blood Causes of inadequate intake of iron in food (infants, vegetarians) Defective absorption, (total or partial gastrectomy, anhydrosis, chronic diarrhea) diseases and fatty diarrhea) prolonged blood loss (gastric ulcer, responsible for the increase in duodenal ulcer other due to chronic bleeding) demand (pregnancy, lactation) storage of RE-based iron content in the cells (chronic or other infections) increase Increased erythrocyte degradation (hemolytic anemia, autoanticorpal anemia
corpal amenia)) Amount of hemoglobin synthesis (pernicious anemia, sideroacretic anemia (sideroac
hrestic amaemia) Acute liver disease (viral hepatitis, toxic hepatitis) Hemochromatosis Hemochromatosis (hemochromatosis) Iron deficiency gradually progresses through various stages before reaching normal anemia. General. Note that low blood iron levels do not necessarily reflect the presence of iron deficiency conditions. Blood iron deviates significantly from normal only when the change in state of saturation of stored iron becomes significant.

Blood iron is a very variable parameter. It varies considerably from day to day and day to day, and the changes are well known.

Various investigators have noted the 24-hour cycle of iron abundance in the blood, which shows that the peak is between 10 am and 10 am
It is between hours and shows a low value late in the evening. Of particular interest is that when working at night, higher values shift in the afternoon, which encodes a cycle of sleep and activity, which seems to reverse the rhythm.

The reference intervals reported in the literature are also different. That is, among other things, normal values are also affected by physiological factors such as age (higher values in neonates and lower values in adults) and gender (slightly higher values in males). .

Blood iron is biologically variable and may increase due to the process of cell necrosis (acute liver disease) and may decrease due to inflammatory conditions (by binding to transferrin) This undermines the diagnostic value of this measurement.

For more information, see, eg, Iron Clinical Significance and Met.
hod of Assfy]] (issued in 1986, Miles Italiana SpA's Ames Div John)
es Division).

The most important problem of iron analysis is that the concentration of the analyte is low, especially in the case of disease. Further, a strong bond between iron and transferrin requires extreme reaction conditions (in an analytical system) to release iron from the carrier protein. Interference caused by, for example, copper is also a significant problem.

The present invention now provides novel compounds, which are very useful as indicator compounds in iron analysis systems. The complex of the compound of the present invention and iron shows not only high sensitivity but also very high stability even in a low pH range. The stability of the iron-indicator complex at low pH is highly desirable. This is because if it is stable, no extreme conditions are required to release the iron from the protein transferrin loading.

The present invention relates to a compound of the general formula (I) Wherein R ₁ represents hydrogen, halogen or alkyl; R ₂ represents C ₁ -C ₄ alkyl, aryl or heteroaryl or substituted C ₁ -C ₄ alkyl, aryl or heteroaryl. It is about.

Preferred compounds are those of the formula (I) in which R ₁ represents hydrogen and R ₂ represents C ₁ -C ₄ alkyl, aryl or thienyl, these radicals being C ₁ -C 4. ₄ Compounds which may be substituted by alkyl, hydrogen, halogen or SO ₃ H.

More preferred are compounds of formula (I) in which R ₁ represents hydrogen and R ₂ represents methyl, ethyl, propyl, phenyl, tolyl or thienyl.

In particular, the invention provides that R ₁ represents hydrogen and R ₂ has the formula In the formula, R ¹¹ represents a compound of the formula (I) representing a group represented by hydrogen, methyl or chlorine.

The most preferred compounds are of the formula It is a compound shown by these.

The invention further relates to an analytical system for detecting or quantifying iron in a sample, comprising one of these compounds.

In a preferred embodiment, the iron analysis system further comprises the formula And a reducing compound represented by the formula:

The invention therefore also relates to the use of this compound for the analysis of iron. This quinoline compound was found to be very useful for the reduction of Fe ⁺⁺⁺ to Fe ⁺⁺ required in iron analysis systems. Although ascorbic acid and other reducing agents known in the art can also be used with this new indicator of the present invention, the above quinoline compounds are preferred for reduction purposes. Therefore, the present invention also provides an analytical system for the determination of iron as a reducing agent.
-Hydroxy-1,2,3,4-tetrahydro-benzo (h)
It also relates to the use of quinolines.

The iron analysis system can further include substances that do not react with, for example, buffers, wetting agents, stabilizers, and the like.

Reagent combinations can be prepared from the compounds described above. The reagent composition can be in the form of a solution,
Alternatively, they can be in powder form, in tablet form, or in lyophilized form. The reagent composition (if not already in solution form) is dissolved in water or another suitable solvent to prepare a reagent solution. If the combination of reagents consists of the individual components, they should be mixed together. After mixing the sample (eg, blood, serum, plasma, or urine) with an aliquot of the reagent mixture, the resulting color is measured with a photometer and determined from the molar extinction coefficient and the volume of reagent and sample added or a standard aqueous solution of iron. Is calculated from

The iron analyzer can also be impregnated with a buffer or with an appropriate wetting agent and activator and other auxiliaries into an absorbent reagent carrier such as iron or fleece. For this purpose, one or more impregnating solutions can be prepared in the form of aqueous solutions, organic solutions or mixed solutions, depending on how the reagents or auxiliaries dissolve. An absorbent or swellable carrier, preferably filter paper or glass or plastic absorbent fleece, is impregnated or sprayed with these solutions. The carrier is then dried. The reagent carrier thus prepared is also used as a rapid diagnostic tool for the direct determination of the content of an analyte in a liquid (eg in a body fluid such as blood, urine or saliva, eg in food such as juice or milk). be able to. These liquids are applied directly to the reagent carrier or are slightly immersed in the body fluid column. A semi-quantitative analysis is possible by comparing the comparative standard color with the color thus formed. Quantitative evaluation can be performed by reflection spectroscopy.

It is also possible to introduce the test agents according to the invention into a carrier matrix prepared from a casting solution. Possible examples are plastics such as cellulose, cellulose derivative gelatin, gelatin derivatives or polyurethanes and polyacrylamide. It is also advantageous here to add the test agent and, if useful, other necessary reagents directly to the casting solution. This means that a test device consisting of a carrier and a reagent can be manufactured in one operation.

The reagents described above can be prepared by eluting the above-mentioned reagents from the absorbent carrier with water or a buffer solution or a condensate, and this can be used as an analytical control in a spectrometer cell as described above. Substances or enzymes can be measured.

Wetting agents are in particular anionic and cationic, nonionic or amphoteric wetting agents.

Other auxiliaries which may be suitable are the usual thickeners, solubilizers, emulsifiers, optical brighteners, contrast agents, as known in the corresponding test methods using other chromogens. It is like a medium.

The production method of the compound of the present invention can be illustrated by the following examples.

The necessary starting materials are described in the literature [eg: Acta . Chem . Scan
. d, 23, 1087 et seq. (1969); J. Amer . Chem . So
s ., 75 , p. 1115 (1953); Organikum, Organish, Chemis
ches Grundpraktikum, known from page 325 onwards (1970).

PREPARATION EXAMPLES Example 1 6.44 g of 3- (2-pyridyl) -5,6-bis (2-thienyl) -1,2,4-triazine were placed at 0 ° C. in 25 ml of fuming sulfuric acid having a titer of 25%. . The resulting reaction mixture was allowed to return to room temperature and stirred for another 24 hours. Next, the sulfonated mixture was poured into about 100 g of ice, equilibrated with a small amount of NaOH, and the precipitate was filtered under reduced pressure. 8.4 g of a yellow powder was isolated, which was mixed with iron (II) ions in water and blue (λ _max
= 593 nm, C = 34000). NMR spectrum reveals that this powder is one of the following possible isomers: Reference Example 1 3- (2-pyridyl) -5,6 required in Example 1
-Bis (2-thienyl) -1,2,4-triazine was prepared as follows.

That is, 13.6 g picolinamidrazone and 22.2 g tenyl were stirred in 125 ml ethanol at room temperature. Two
After 4 hours, the reaction mixture was concentrated on a rotary evaporator and the residue was recrystallized from absolute alcohol. C-
The 13 NMR data is as follows.

C-1: 150.40D C-2: 125.33D C-3: 139.01D C-4: 124.04D C-5: 160.04D C-6: 152.63S C-7: 149.54 ^a S C-8: *** ^{* a .S C-9: 136.89S} C-10: 131.73D C-11: 129.60D C-12: 128.83D C-13: 137.11D C-14: 128.27D C-15: 129.81D C-16: 132.32D Reference Example 2 284 g of phosphorus pentoxide was stirred in a mixture of 2500 ml of toluene and 250 ml of thiophene. Next, 300 g of tolyl acetic acid was added in small portions at 80 ° C. Subsequently, the resulting mixture was stirred for 5 hours and then poured into the table. The obtained organic layer was separated, dried and concentrated by a rotary evaporator. The residue was recrystallized from aqueous ethanol to give 326 g of the following formula JR = 1660 cm ^-1 (C = 0) was obtained.

Reference Example 3 27.75 g of selenium dioxide was suspended in a mixture of 250 ml of dioxane and 20 ml of water. Thionyl ketone of Reference Example 2
54.0 g was added to this suspension, and the resulting mixture was heated under reflux for 6 hours and then filtered under reduced pressure to remove the residue, and the obtained reaction mixture was concentrated on a rotary evaporator.

49.2 g of a brown oil were obtained which was subjected to the next step without purification.

Reference Example 4 23.0 g of the diketone prepared in Reference Example 3 was heated under reflux with 13.6 g of picolinamidrazone in 100 ml of ethanol. The triazine formed had already crystallized under boiling heat. After 1 hour, the mixture was cooled and filtered under reduced pressure.

33.9 g of a yellow powder with a melting point of 191 ° C. were isolated. From the spectrum, the product cannot be clearly stated as being one or the other of the two isomers shown below.

Reference Example 5 The triazine of Reference Example 4 was reacted with fuming sulfuric acid having a titer of 25% according to the method described in Example 1 to obtain a monosulfonated compound.
One.

Calculated value; C, 52.77; H, 3.03; N.12.96; O, 11.1; S, 14.83; Na, 5.31. Actual value; C, 52.3; H, 3.1; N. 13.0; S, 15.0. Example 2 6.44 g of 3- (2-pyridyl) -5,6-bis (2-thienyl) -1,2,4-triazine in 50 ml of sulfonated monohydrate at 50 ° C. for 7 hours, resulting in a mixture. Was treated as described in Example 1 to obtain 3.2 g of the following compound having a melting point of 250 ° C. or higher.

Reference Example 6 When pyridinylthiophene is reacted with picolinamidrazone as described in Reference Example 4, one of the two possible triazines shown below has a yield of 80% [JR; 1385 cm
^-1 obtained in (-CH _3)].

Reference Example 7 The triazine derivative of Reference Example 6 was sulfonated in monohydrate at a temperature of 50 ° C. After work-up, a yellow powder corresponding to the following formula 1 was obtained in a yield of 65%.

Elemental analysis Calculated value: C, 43.82; H.2.55; N. 15.72; O, 13.47; S, 17.99; Na, 6.45. Actual value; H, 44.00; H, 2.45; S, 18.2. Test example Next iron test In Example 2, the compound of Example 1 was used as an indicator.

Principle of Test Iron in human serum is released from transferrin, which is a carrier protein, in a suitable medium, and at the same time is reduced to divalent iron by a reducing agent. The chelate of the indicator and the divalent iron ion forms a stable blue complex, the absorbance of which at 593 nm (nanometer), read by a spectrophotometer, is proportional to the iron content. Deproteinization is not required. Sample blanks are required to ensure serum matrix effects.

Materials and Methods Experiments (optimization of the method, linearity, comparative studies, etc.) were performed according to the following specifications.

Sample: (Heparin-added human plasma or human serum (as-is or iron ion-added) obtained from daily work in a hospital was used. It was prepared by dissolving NBS agent code 937) and diluting it with distilled water to an appropriate concentration.

Apparatus: Double beam spectrophotometer (Model Lambda 5,
Perkin Elmer).

Materials: The iron indicator was the compound of Example 1 and all other compounds were reagent grade materials. The solution actually used contains a buffer, a reducing agent, a thiourea (to suppress possible copper interference) and an indicator compound. A solution containing no indicator compound was also prepared for the sample blank. For the comparison test, a SERA-PAK Iron kit (by Arenes, Miles Italiana SpA Ferene-S) was used.

Test operation Wavelength: 593 nm (570 to 610) Cuvette: Optical path length 1 cm Temperature: Room temperature Reading: For standard samples and samples for reagent blanks; For sample blanks for distilled water Pipetting into test tubes: Mix and leave at room temperature for 5 minutes. Absorbance of sample blank (Asb) against distilled water and sample (A
Read absorbance of s) and standard (Ast).

Optimization studies Optimizing pH Starting with a formulation containing the following components:

Indicator 3.5 mmol / L thiourea 63 mmol / L Ascorbic acid 10 mmol / L Absorber 180 mmol / L; pH range 0.5-5.0 Concentration of 200, 500, and 100 μg / dl of iron ion aqueous solution The effect of pH on the iron test was investigated using two human plasma samples containing 300 μg / dl iron. Different types of buffers were used to cover a given pH range.

KCl / HCl for pH 0.5-1.0-1.5-2.0 Citric acid / NaOH for pH 2.0-2.5-3.5 Acetic acid / NaOH for pH 3.0-4.5-5.0 Color development monitored at 593 nm, 5 minutes at room temperature After (end point of reaction)
The absorbance was measured (see Fig. 1). The average absorbance for three aqueous solutions of iron and two plasma materials was calculated and plotted against pH (Fig. 1).

From these data, the indicator compounds of the present invention can be used at a pH of 1 or higher, preferably about 1, to promote the release of iron from transferrin.

Selection of Buffer A compound capable of providing a buffer solution at pH 1 was selected. For example, using KCl / HCl or citric acid or malonic acid as a buffer, a test was conducted at pH 1.0, and the iron content was 0.3 mol / mol.
L aqueous solution and human plasma were used. No difference was observed in absorbance response and reaction time. All compounds tested were found to have the same buffer capacity for human serum.

Ascorbic acid is a commonly used reducing agent for reducing ferric ions, but unfortunately, this compound, when brought into solution, only It is only stable for 3 hours. Thus, generally, in commercial iron kits, ascorbic acid is supplied in powder form for manual addition to the test solution (see Sera-Pak iron kit). In order to obtain a ready-to-use solution, studies were conducted to find a more stable and suitable reducing agent.

3-hydroxy-1,2,3,4-tetrahydro-benzo (h) quinoline (HTBQ): Has been found to be very suitable for reducing trivalent iron ions to divalent ions in an acid complex, ascorbic acid, and promoting the release of iron from transferrin.

 Starting with a formulation containing the following components: HCl / KCl buffer, pH 1.0; 100 mmol / L indicator 3.5 mmol / L thiourea 63 mmol / L.

Add HTBQ at a concentration ranging from 0 to 25 mmol / L, and after 5 minutes of reaction
The absorbance response to 593 nm was stored using an aqueous solution of iron and two human plasma materials. The data in Fig.2 is
A minimum amount of 5-10 mmol / L HTBQ is required, indicating that a concentration of about 10 mmol / L is preferred.

Indicator optimization Starting from a formulation containing HCl / KCl buffer, pH 1.0 200 mmol / L thiourea 63 mmol / L HTBQ 20 mmol / L.

To this, an indicator was added in a concentration range of 0.5 to 10 mmol / L, and 5
The absorbance response at 593 nm was monitored using an aqueous solution of iron and two different human plasma samples after a one minute reaction. The data in FIG. 3 shows that a minimum of 2.5-3 mmol / L of indicator is required, and a concentration of about 3.5 mmol / L is preferred.

 From the optimization studies performed, we found the following:

1. pH: This system operates in the pH range of 1-5, but pH = 1
Is preferred to promote dissociation of iron from transferrin. If a pH above 3.0 is selected, it is preferred to add a surfactant to the test solution to prevent possible clouding of the sample, but for this purpose Triton X-100 or Tww
een10 can be used at a concentration of 0.5%. More than 5
The pH has not been tested, but the system appears to be operable with the appropriate components to dissociate iron from transferrin.

2. Buffers: Various types of compounds can be used (eg, citric acid, malonic acid, HCl / KCl).

3. Molarity: less than 400 mmol / L, preferably about 200 mmol / L, to prevent possible clouding of human samples
Is preferred, but with the use of suitable surfactants, moral concentrations above 400 mmol / L can be used.

4. Reducing agent: It is convenient to replace ascorbic acid with HTBQ. Concentrations above 5 mmol / L are suggested, but about 20 m
A concentration of mol / L is preferred.

5. Indicator compound: a concentration of more than 2.5 mmol / L is suggested, but a concentration of about 3.5 mmol / L is preferred.

6. Thiourea: If a formulation with pH 1.0 is used, this component is not necessary and its use can be avoided. this
If the pH value is exceeded, 63 to prevent copper interference
A concentration of mmol / L is satisfactory (data not shown).

Confirmation of performance The performance was confirmed using the test procedure reported previously and the following components: HCl / KCl interference solution, pH 1.0 200 mmol / L Indicator 3.5 mmol / L Formulation containing HTBQ 20 mmol / L .

Test of linearity A standard aqueous solution of trivalent iron ion prepared by dissolving metallic iron (NBS material) in nitric acid and diluted to an appropriate concentration with distilled water was tested three times. Fig. 4 shows the directness up to at least 100 μg / dl iron.

Comparative studies Comparative tests were performed using the Sera-Pak iron kit and the formulation of the present invention. Twenty-five heparinized human plasma were used as samples.

Fig. 5 shows the results obtained by statistical refinement by the linear method. The correlation between the two methods is very good.

[Brief description of the drawings]

FIG. 1 is a graph showing the relationship between the pH and the average absorbance at 593 nm of an aqueous iron solution and a plasma sample. FIG. 2 is a graph showing the relationship between the amount of HTBQ (reducing agent) added and the absorbance (593 nm) of the measurement sample. Fig. 3 shows the concentration of the indicator and the absorbance of the measurement sample (593n
It is a graph which shows the relationship of m). FIG. 4 is a graph showing the relationship between the iron content and the absorbance (593 nm). FIG. 5 is a graph showing the correlation when using SERA-PAK and the indicator composition of the present invention. FIG. 6 and FIG. 7 are graphs showing the relationship between wavelength and absorbance.

 ──────────────────────────────────────────────────続 き Continuing on the front page (72) Inventor Herbert Hugl Dee-5060 Berg Gladbach 2, Gemarkenweg 9 (72) Inventor Klaus Wehring Dee-5060 Wupperol, Germany 1. Am Rohm 121 (56) References JP-A-57-5680 (JP, A)

Claims (5)

(57) [Claims] 1. The compound of the general formula (I) (Wherein R ¹ represents hydrogen, halogen or C ₁ -C ₄ alkyl) or a salt thereof. 2. The compound according to claim 1, wherein R ¹ represents hydrogen, or a salt thereof. 3. The expression Or a salt thereof. 4. An analysis reagent for detecting or quantitatively analyzing iron in a sample, comprising the compound according to claim 1 or a salt thereof. 5. The method according to claim 1, wherein The analytical reagent according to claim 4, comprising a compound represented by the formula: